Severe streptococcal infections account for more than 500,000 deaths globally per year. The rapid emergence of multi-drug resistant bacterial pathogens, and the lack of new antibiotics in pharmaceutical development, represents a huge public health problem. Streptokinase (SK), a major streptococcal virulence factor, targets human fibrinolysis by activating plasminogen (Pg) to the proteinase, plasmin (Pm). By mechanisms that are not understood, Pm degrades protective fibrin (Fbn) barriers generated by the host response. Along with another virulence factor, PAM, SK produces, by an unknown mechanism, Pm bound to the bacterial surface, allowing Pm-coated bacteria to spread through tissues. Background - We proposed a unified mechanism of SK-Pg activation initiated by SK binding and inducing conformational activation of the Pg zymogen by the NH2-terminal insertion mechanism. A second Pg molecule binds to the SK7Pg* catalytic complex as a specific substrate, which is cleaved to Pm in an initial, triggering catalytic cycle. Pg is displaced from SK7Pg* by the high affinity binding of Pm produced, yielding the SK7Pm catalytic complex that binds Pg as a substrate, initiating the second, propagation cycle that converts the remaining Pg to Pm. Broad Goals - Biochemical and biophysical studies of Pg activation by SK are proposed with the goal of understanding the molecular mechanism by which SK subverts human fibrinolysis to promote severe streptococcal diseases, with the ultimate purpose of developing new mechanism-based drugs. The investigation addresses hypotheses to fill major gaps in the understanding of the infection mechanisms, and evaluation of novel therapeutic approaches. Experimental approaches include enzyme kinetics, rapid-reaction kinetics, equilibrium binding, mutagenesis, and collaborative electron microscopy, crystallography, and in vivo models of infection. Aim 1: To define the role of [Glu]Pg conformational changes in its binding to SK in the catalytic and substrate modes, and the mechanism of substrate/product recognition for [Glu]Pg, [Lys]Pg, and Pm. Aim 2: To define in rapid-reaction kinetic studies the pathway of molecular events in the mechanism of SK-induced conformational activation of [Glu]Pg and [Lys]Pg, and the mechanism of [Glu]Pg/[Lys]Pg substrate recognition by the SK7Pg*/Pm catalytic complexes. Aim 3: To determine the role of inter-domain flexibility of SK bound to Pg and Pm in dictating SK function, and to evaluate antibodies against the flexible segments linking SK domains as inhibitors for potential use in combating streptococcal diseases. Aim 4: To elucidate the mechanisms by which Fbg, Fbn, and PAM regulate SK-initiated Pg activation that underlie their roles in invasive streptococcal infections, and to evaluate an anti-substrate Pg recognition antibody inhibitor. Aim 5: To define the mechanism of skizzle (SkzL) as a novel cofactor of tPA-catalyzed Pg activation, and to evaluate its potential role as pathogenicity factor in Streptococcus agalactiae infections.